Year after year, microfluidic devices appear more clearly as a valuable alternative to conventional systems for numerous applications. Several commercial systems are already on the market, and their potential applications increase steadily. There are, however, a number of application that seem to resist this trend. For the detection of point mutations in DNA by heteroduplex analysis, for instance, earlier studies demonstrated that the resolution depends critically on the length of the capillary used in separation, because the difference in mobility between two duplex DNA fragments with the same length and a single by mismatch is very minute. Recently, innovative matrices could increase this difference, and allow highly reliable separations in bench-top DNA sequencers, but this performance could be achieved in 50 cm long capillaries only. In order to transpose this protocol to microchip format, without compromising the resolution obtained with 50 cm long capillaries, both sharp injection bands and long (10-20 cm) separation channels are required. As recited in Liu, S. et al., (Anal. Chem. 1999, 71, 566-573) another application that requires long channels is DNA sequencing on a chip with high read-lengths. Ramsey, (Anal. Chem. 2003, 75, 3758-3764) disclosed a design, in which they have addressed the problem of fabricating long channels by introducing serpentine or spiral geometries. However, the presence of turns in such geometries introduces band dispersion (“racetrack effect”), as described in Paegel, et al. (Anal. Chem. 2000, 72, 3030-3037(.
Koutney, et al, (Anal. Chem. 2000, 72, 3388-3391) describe a glass-based DNA sequencing chip with a 40 cm long straight separation channel, which solves this problem of channel length. However, this device requires complex lithographic steps (e.g. specialized spin-coating, direct UV-laser writing, wet chemical etching and thermal bonding). This makes the construction and operation costs of such chips extremely high. Also these large microchannel arrays are difficult to manipulate.
For lab-on-chips to find their way into routine clinical analysis, the microdevices must be inexpensive, disposable and easy to fabricate, while retaining the high resolution performances of state-of the art devices based on long glass capillaries. There is thus a strong need of proposing a low cost process, allowing the fabrication of microfluidic systems comprising microchannel networks, involving at least one long microchannel, typically of length 10 cm and larger. There is also a need to prepare robust, easy to manipulate and compact devices comprising microchannel networks involving at least one long microchannel without sharp turns detrimental to resolution.
To fulfil the requirement of low cost and ease of fabrication, polymers and plastics are increasingly replacing traditional microfluidic substrates like silicon and glass in diagnostic applications. Some examples of such devices are reviewed in Becker, et al., (Talanta 2002, 56, 267-287). Due to the same reason, standard lithographic fabrication of devices is giving way to “soft lithography” or replication-based methods, (e.g. mold casting, nano-imprinting, thermoforming, hot-embossing, etc.) in which a large number of devices can be replicated in a short time from a single master. The company Gyros, for instance, discloses in U.S. Pat. No. 6,126,765 and U.S. Pat. No. 6,620,478 devices able to accommodate straight channels about 5 cm long by arranging all the channels along the radius and transporting liquids using centrifugal flow, and methods of fabrication for such devices. These devices are fabricated by injection molding of thermoplastics in a format compatible with that of optical CDs and DVDs, thus making them very inexpensive in mass production by injection molding. The length available in a CD format is still insufficient for high resolutions sequencing, however. Laboratory-scale replication methods (e.g. mold casting of elastomers or hot-embossing of thermoplastics using a press) are not convenient either for fabricating long straight channels.
Hence it is important to develop new strategies for fabrication of long channels in plastic substrates, compatible both with laboratory scale and industrialisation.
Another emerging area of micro fluidics is the fabrication of devices in thin polymer substrates to yield “flexible” chips. This technology has the potential of allowing low-cost fabrication in a lamination process, as recited e.g. in U.S. Pat. No. 6,761,962 to Bentsen, or in US 2005/0089449 to Polwart. In contrast with hot embossing or injection molding, in which the substrate is fully enclosed in a container, and can thus be raised above its glass transition for an arbitrary length of time in order to allow for an accurate reproduction of the microstructure of the mold, continuous microfabrication processes based on lamination raise specific and difficult problems. As a solution to these problems, Bentsen, U.S. Pat. No. 6,761,962, proposed to deliver the substrate as a liquid that is dye casted onto a supporting layer with a higher glass transition. Once the microstructures imprinted into the substrate, the latter is solidified by crosslinking or cooling. This, however, makes the fabrication complex, and restricts the number of materials that can be used. It also raises problem of adhesion between the substrate and the supporting layer. U.S. Pat. No. 6,761,962 also propose that microstructures be imprinted into a preformed sheet of material by hot embossing on a molding roll. However, a thin flexible substrate cannot be raised globally above its glass transition and kept under tension, since it would deform or even break. No example of microstructures made by this way were presented in the above patent. U.S. Pat. No. 6,838,156 to Neyer, proposes a solution to this problem, consisting in heating the substrate in the vicinity of the microstructures, using high energy radiation to locally heat the master. This requires, however, that the molding device be at least partially transparent to said radiation, which is not convenient for industrial processes. US 2005/0089449 to Polwart, in contrast, use a method based on high pressure plastic film forming. This method, however, is limited to very thin films, thus requiring an additional supporting case, and it is also limited to relatively large microstructures, of order 100 μm. Finally, in J. Micromech. Microeng. 2006, 16, 113-121, Abgrall, et al. demonstrates the fabrication of flexible 3D microfluidic networks in the photosensitive resin SU-8. However, this photocurable resin requires serial processing and relatively long curing and developing, rendering it inadequate for cost-effective mass production.
It would thus be very beneficial to propose new methods, able to facilitate the accurate reproduction of microstructures or nanostructures in sheetlike substrates, and in particular in flexible, thin ones.
The choice of the right material for the development of high resolution microfluidic DNA electrophoresis devices is also critical and non trivial, since the chosen material should combine optical qualities approaching those of glass or ideally fused silica, for optimal detection, surface properties avoiding biomolecules adsorption and electroosmosis, and amenability to good replication of micron-sized structures. Polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polycarbonate (PC), polystyrene (PS), polyimide, cyclic olefin copolymer (COC), and polyethylene (PE) are some of the common polymeric materials used to fabricate chips (reviewed in Becker, et al., Talanta 2002, 56, 267-287). Among these, PDMS is the most popular substrate for soft lithography due to its low affinity for biomolecules and cells, transparency in the UV region (which allows integration of optical detection modules) and easy sealing of devices (both reversibly and irreversibly). However, PDMS also suffers from certain disadvantages, such as, swelling in organic solvents (thus limiting the range of microfluidic applications), low mechanical strength (leading to sagging of high-aspect ratio structures in the device) and unstable surface treatments. Oxidized PDMS becomes hydrophobic in air within 30 minutes, thereby not being able to prevent non-specific adsorption of molecules on the surface of the device. Generally speaking, it is very important to be able to achieve a surface treatment of a polymeric material used for the fabrication of a microsystem or of a microfluidic system, in order to bond some reactive species such as biological ligands or enzymes, to prevent unwanted adsorption of species, or to modify the wettability of said surfaces. Numerous methods have been proposed in the art, as reviewed e.g. by Rohr et al. (Adv. Funct. Mat., 2003, 13, 264-270). Hu et al. describes in Anal. Chem. 2002, 74, 4117-4123 a method for grafting polymer onto the polymer PDMS, thanks to UV activation of said PDMS. In this method, the PDMS was contacted with an aqueous solution containing NaIO4, benzyl alcohol, and acrylic monomer, and the polymerization of the acrylic polymer was photoinitiated. This method, however, does not lead to a treatment that is very stable in time, and it leads to imperfect surface treatment. For instance, a good surface treatment with a neutral polymer such as polyacrylamide should lead to an electroosmotic flow as low as 10−5 cm2/Vs, whereas only around 1.10−4 cm2/Vs were achieved with this method. Cyclo-olefin copolymer (COC) is another promising plastic substrate for microfluidic devices due to its chemical resistance to acids, bases and most polar solvents (De Mello, A., Lab Chip 2002, 2, 31N-36N). Cyclo-olefin polymer devices fabricated by different techniques such as injection molding, micromilling, thermal nanoimprint lithography and hot-embossing using a press have been disclosed, e.g. in U.S. Pat. No. 6,787,015 to Lakcritz.
However, due its chemical inertness, this polymer is not easily amenable to surface treatment and bonding. This is also true for many other polymers interesting for microfluidic systems such as, as an examplary list, polyolefins, fluoropolymers, polyesters, and the like. More generally, bonding materials presenting microstructures or nanostructures without altering these structures remains a challenge.
It would thus be useful to propose methods able to induce an efficient surface treatment onto a wide variety of polymers, in particular but not exclusively, chemically inert, difficult to functionalize or elastomeric ones.
Another challenge in the fabrication of embedded microstructures such as microchannels, is the closing of microchannels. Typically, microchannels are fabricated in two steps. In a first step, recessed microstructures corresponding to the microchannels are prepared in one substrate, by a technique known by those skilled in the art such as casting, photolithography, hot embossing, injection molding, micromilling, photoablation, plasma ablation, powder blasting, and the like. In a second step, a second substrate, which may optionally also bear microstructures, is bonded onto the first substrate to close the channel. This bonding can be achieved by chemical means or by physical means. A widely used means for bonding two substrates in order to create embedded microchannels consists in using an intermediate adhesive layer. Optionally, this intermediate layer can be of the “stencil” type, i.e. it may carry holes or slots crossing the whole layer, that will constitute the wanted microchannels after bonding of one substrate to each of the sides of said stencil. US 2005-0205136 A1 to Freeman, for instance, proposes such an approach. A disadvantage of this method, however, is that the lateral walls of the microchannel are of a different chemical nature as the top and bottom walls. A widely used way to bond two polymeric substrates is thermal bonding. In this case, it was proposed to introduce between the two substrates to be bonded a layer of “hot melt” type, or more generally of a thermoplastic material with a deformation temperature smaller than that of the surfaces to bond, as disclosed e.g. in U.S. Pat. No. 6,126,765 to Ohman et al. In that case, however, the bottom and top surfaces of the microchannel have different chemical natures. U.S. Pat. No. 6,503,359 to Virtanen proposes another method using chemical bonding. U.S. Pat. No. 5,932,799 to Moles proposes a method specific for systems made in polyimide, using so called “self-bonding” polyimide. This polyimide contains additives such as Sn that stimulate thermally excited chemical crosslinking between the surfaces to be bonded. All of the above methods for bonding two substrates in order to create an embedded microchannel, however, share the inconvenient, that they cannot lead to a microchannel with uniform surface properties around its perimeter. This is very detrimental to numerous applications, in particular those involving the transport in the microchannels, of species that tend to adsorb on the microchannel surfaces. This is also very disadvantageous to electrophoretic separation methods, or more generally to electrokinetic transport, because differences in surface properties lead to inhomogeneous electroosmosis, which in turn lead to dispersion.
US 2003/0150555 A1 to Gandhi proposes an other method, in which one of the two substrates to be bonded have different glass transitions. In this case, one can generally achieve surface properties that are relatively uniform, because the difference in glass transition can be achieved by changes in molecular weight, which do not change significantly the surface properties. However, in this method, in contrast with e.g. U.S. Pat. No. 6,838,156, thermal bonding implies that one of the polymer substrates to be bonded is brought above its glass transition. It is thus very difficult to keep microstructures intact: if the substrate carrying microstructures is the one with the lowest glass transition, the structures will tend to collapse during bonding. In contrast, if the layer with the lowest glass transition is a planar cover substrate, it will tend to flow into the microstructures, and also lead to an alteration of the wanted microchannel characteristics. This problem is particularly serious for the fabrication of thin-film systems. For instance, sub-micron features have been reported to be patterned on spin-coated polystyrene films which could be peeled off and folded, but without being sealed (Hazarika et al. Lab Chip 2003, 3, 128-131). The preparation of sealed, thin microfluidic systems is disclosed in US 2005-0089449, but the microstructures prepared were rather large, and the systems prepared this way do not present uniform surface properties all around the microchannel perimeter.
So, there is a strong need to develop low-cost, high throughput methods for preparing embedded microchannels or microstructures with substantially uniform surface properties.
It is thus an object of the present invention, to propose low-cost, robust and flexible microfluidic systems comprising embedded microstructures such as one or several long microchannels.
It is another object of the invention to achieve such goal without introducing along said microchannel sharp turns.
It is also an object of the invention to propose improved methods suitable for fabricating microsystems with embedded microchannels or microstructures at a low cost.
In particular, it is a further object of the invention to propose improved methods for treating the surface of a sheetlike substrate.
As one of its advantages, the invention allows the easy fabrication of microsystems that would have been either impossible or very difficult to produce with prior art. Thus, it is also an object of the invention to propose large, flexible, integral microsystems comprising at least one embedded microchannels network, wherein said network involves at least one long microchannel.